Aerobic Ammonium-oxidizing Bacteria Research Articles

Rapid start-up and microbial characteristics of partial nitrification granular sludge treating domestic sewage at room temperature

The successful suppression of nitrite-oxidizing bacteria in the partial nitrification (PN) stage was the main challenge for the application of autotrophic nitrogen removal process treating mainstream sewage. In this study, two identical PN granular reactors (P1 and P2) were rapid started-up using the simultaneous PN and granulation strategy, for treating the domestic sewage. P1 was seeded with 30% PN granular sludge to induce nucleation, in which the granule size achieved to more than 400μm in 12d, with ammonia oxidation rate and nitrite accumulation rate of 80% and 95%, respectively, while P2 realized granulation in 42d. The presence of organic matters and specific structure of granules were profitable for the stability of PN for treating sewage with low ammonia. High-throughput pyrosequencing results indicated the biodiversity of both reactors decreased after start-up, and Nitrosomonas was the predominant specie of aerobic ammonia-oxidizing bacteria in PN granular sludge.

Microbial community of nitrogen-converting bacteria in anammox granular sludge

The nitrogen removal pathways in Anaerobic ammonium oxidation (anammox) process where anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite directly to dinitrogen gas under anoxic conditions were investigated in this study. For this propose, microbial community of anammox granules from the lab-scale and full scale reactors were analyzed according to morphology, diversity and activity. The microbial diversity of anaerobic ammonia oxidizing bacteria (anAOB), heterotrophic denitrifying bacteria (HDB) and aerobic ammonium-oxidizing bacteria (AerAOB) was examined by clone libraries. The abundance of AnAOB and HDB was quantified by real time PCR (qPCR). The activities of partial nitrification, denitrification and anammox were estimated with batch tests and respirometry. The fluorescence in situ hybridization (FISH) showed an outer layer dominated by AnAOB whereas the core had a very low cell density with no AnAOB. The denaturing gradient gel electrophoresis (DGGE) results show the existence of genes related to the nitrogen-compounds-converting bacteria from AnAOB (16S rRNA), HDB (nirS) and AerAOB (amoA). In the context of phylogenetical analysis 35 AnAOB clones were analyzed belonging to the genus Candidatus Brocadia (2 Operational taxonomic units). On the other hand, amongst the 26 sequenced HDB clones had a high richness of different nirS genes (13 OTUs). In 38 clones of the amoA gene 7 OTUs were detected. The specific activity of the AnAOB was 3.7–5.6 mg N g−1 VSS h−1. Anammox was the main pathway for the ammonia degradation. The average HDB activity ranged around 0.9 mg N g−1 VSS h−1. With nitrate as a sole substrate for the HDB the average activity for the HDB without external carbon source was around 1.5 mg NO3–N g−1 VSS h−1. The activity of the AerAOB measured by respirometry test reached only 0.05 mg N g−1 h−1.

Effect of COD/N ratio on nitrogen removal and microbial communities of CANON process in membrane bioreactors

In this study, the effect of COD/N ratio on completely autotrophic nitrogen removal over nitrite (CANON) process was investigated in five identical membrane bioreactors. The five reactors were simultaneously seeded for 1L CANON sludge and be operated for more than two months under same conditions, with influent COD/N ratio of 0, 0.5, 1, 2 and 4, respectively. DGGE was used to analyze the microbial communities of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AAOB) in five reactors. Results revealed the harmonious work of CANON and denitrification with low COD concentration, whereas too high COD concentration suppressed both AOB and AAOB. AOB and AAOB biodiversity both decreased with COD increasing, which then led to worse nitrogen removal. The suppressing threshold of COD/N ratio for CANON was 1.7. CANON was feasible for treating low COD/N sewage, while the high sewage should be converted by anaerobic biogas producing process in advance.

Performance of one-stage autotrophic nitrogen removal in a biofilm reactor with low C/N ratio

Wastewater with C/N ratios ranging from 1.00 to 0.33 caused by a gradual increase in influent concentration was used to evaluate the performance of the one-stage nitrogen removal process in a biofilm reactor. The system was operated for 197 days under chemical oxygen demand (COD) concentration of 250 mg L−1 and influent concentrations ranging from 250 to 750 mg L−1. The effects of the C/N ratio and dissolved oxygen (DO) on nitrogen removal were evaluated at different influent C/N ratios and DO concentrations, respectively. The microbial composition of the system was examined by scanning electron microscopy and polymerase chain reaction-denaturing gradient gel electrophoresis, and the relative contribution of anaerobic ammonium oxidation (ANAMMOX) to nitrogen removal was assessed by calculating the average rates of ANAMMOX and denitrification in batch experiments. Results showed that the removal efficiencies of total nitrogen (TN), and COD were 74–97%, 75–99% and 64–97%, respectively. The C/N ratio had a significant influence on nitrogen removal efficiency when it was decreased from 1.00 to 0.70, but no significant change was observed when it was reduced from 0.70 to 0.33. DO also correlated with the concentration in the influent, and 3.0 mg L−1 was found to be a suitable concentration for the influent concentration of 450 ± 5 mg L−1. Analysis of microbial composition of the system revealed that biofilm and activated sludge were mainly composed of aerobic ammonium-oxidizing bacteria, anaerobic ammonium-oxidizing bacteria (AnAOB) and denitrifying bacteria. Activity tests suggested that AnAOB played an important role in the one-stage autotrophic nitrogen removal process, contributing to about 52.7% of total TN removal via ANAMMOX.

Combination of upflow anaerobic sludge blanket (UASB) reactor and partial nitritation/anammox moving bed biofilm reactor (MBBR) for municipal wastewater treatment

In this study the combination of an upflow anaerobic sludge blanket (UASB) reactor and a deammonification moving bed biofilm reactor (MBBR) for mainstream wastewater treatment was tested. The competition between aerobic ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) was studied during a 5months period of transition from reject water to mainstream wastewater followed by a 16months period of mainstream wastewater treatment. The decrease of influent ammonium concentration led to a wash-out of suspended biomass which had a major contribution to nitrite production. Influence of a dissolved oxygen concentration and a transient anoxia mechanism of NOB suppression were studied. It was shown that anoxic phase duration has no effect on NOB metabolism recovery and oxygen diffusion rather than affinities of AOB and NOB to oxygen determine the rate of nitrogen conversion in a biofilm system. Anammox activity remained on the level comparable to reject water treatment systems.

Effect of trace hydrazine addition on the functional bacterial community of a sequencing batch reactor performing completely autotrophic nitrogen removal over nitrite

A sequencing batch reactor (SBR) was conducted to perform completely autotrophic nitrogen removal over nitrite (CANON). The effect of long-term trace N2H4 addition on ammonium oxidizing bacteria (AOB) and anaerobic AOB (AnAOB) in the CANON system was investigated. AOB and AnAOB primarily related to Nitrosococcus, Nitrosomonas and Candidatus scalindua, respectively. Before and after trace N2H4 addition, the estimates of AOB population decreased from 1.03×107 to 6.25×104copies/g (dry sludge), but that of AnAOB increased from 3.14×109 to 5.86×1010copies/g (dry sludge). Despite there was a partially negative impact on AOB growth, the trace N2H4 addition exerted a stronger inhibition on nitrite oxidizing bacteria (NOB) and promoted AnAOB growth, which improved the nitrogen removal of the CANON system. Sludge granules enriched under long-term trace N2H4 addition were spherical and ellipsoidal, and the aerobic AOB were mainly located on the outer layers while AnAOB occupied most of the interior parts.

Performance and influence factors of completely autotrophic nitrogen removal over nitrite (CANON) process in a biofilter packed with volcanic rocks

Completely autotrophic nitrogen removal over nitrite (CANON) process was considered as one of the most efficient and economical nitrogen removal processes, which was suitable for treating wastewater with low ratio of carbon to nitrogen. In this study, an enlarging start-up strategy for CANON process was proposed, and a 40-L CANON reactor was successfully started by seeding 2-L mature biofilm containing both aerobic ammonia-oxidizing bacteria (AerAOB) and anaerobic ammonia-oxidizing bacteria (AnAOB). The effects of dissolved oxygen (DO), ammonia loading rate and the ratio of air inflow to water inflow (Qair/Qwater) on nitrogen removal performance were investigated. The distribution of AerAOB and AnAOB was analysed using fluorescence in situ hybridization (FISH) technique. The system reached a maximum NRR of 3.11 kg N m−3 d−1 with a removal efficiency of 89.5%, and the average value in steady state was 2.42±0.26 and (83.07 ± 6.89)%, respectively. Analysis of influence factors showed the important role of high DO (around 5 mg L−1), for the high-rate nitrogen removal, and the Qair/Qwater should be controlled at 28–40 for stable operation. FISH results suggested that AerAOB and AnAOB predominated in the reactor, with proportions of 46.8% and 39.3%, respectively. This study demonstrated that the biofilter operated with high effluent DO was a feasible setup for CANON process.

Nitrogen removal and microbial characteristics in CANON biofilters fed with different ammonia levels

The nitrogen removal performance and microbial characteristics of four completely autotrophic nitrogen removal over nitrite (CANON) biofilters were investigated. These four reactors were simultaneously seeded from a stable CANON biofilter with a seeding ratio of 1:1, which were fed with different ammonia levels. Results suggested that with the ammonia of 200–400mgL−1, aerobic ammonia-oxidizing bacteria (AerAOB) and anaerobic ammonia-oxidizing bacteria (AnAOB) could perform harmonious work. The bioactivity and population of the two groups of bacteria were both high, which then resulted in excellent nitrogen removal, while too low or too high ammonia would both lead to worse performance. When ammonia was too high, the bioactivity, biodiversity and population of AerAOB all decreased and then resulted in the lowest nitrogen removal. Nitrosomonas and Candidatus Brocadia were detected as predominant functional microbes in all the four reactors. Finally, strategies for treating sewage with different ammonia levels were proposed.

Distribution patterns of ammonia-oxidizing bacteria and anammox bacteria in the freshwater marsh of Honghe wetland in Northeast China.

Community characteristics of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria in Honghe freshwater marsh, a Ramsar-designated wetland in Northeast China, were analyzed in this study. Samples were collected from surface and low layers of sediments in the Experimental, Buffer, and Core Zones in the reserve. Community structures of AOB were investigated using both 16S rRNA and amoA (encoding for the α-subunit of the ammonia monooxygenase) genes. Majority of both 16S rRNA and amoA gene-PCR amplified sequences obtained from the samples in the three zones affiliated with Nitrosospira, which agreed with other wetland studies. A relatively high richness of β-AOB amoA gene detected in the freshwater marsh might suggest minimal external pressure was experienced, providing a suitable habitat for β-AOB communities. Anammox bacteria communities were assessed using both 16S rRNA and hzo (encoding for hydrazine oxidoreductase) genes. However, PCR amplification of the hzo gene in all samples failed, suggesting that the utilization of hzo biomarker for detecting anammox bacteria in freshwater marsh might have serious limitations. Results with 16S rRNA gene showed that Candidatus Kuenenia was detected in only the Experimental Zone, whereas Ca. Scalindua including different lineages was observed in both the Buffer and Experimental Zones but not the Core Zone. These results indicated that both AOB and anammox bacteria have specific distribution patterns in the ecosystem corresponding to the extent of anthropogenic impact.

Microbial characteristics and nitrogen removal of simultaneous partial nitrification, anammox and denitrification (SNAD) process treating low C/N ratio sewage

Simultaneous partial nitrification, anammox and denitrification (SNAD) process was successfully realized for treating low C/N ratio sewage, nitrogen and COD removal achieved to 3.26kgm−3d−1, 81%, respectively. The nitrogen removal performance, microbial community and distribution of the functional microorganisms were investigated. Results suggested that the presence of COD performed activity inhibition on both aerobic ammonia-oxidizing bacteria (AerAOB) and anaerobic ammonia-oxidizing bacteria (AnAOB), and led to the number decreasing of both AerAOB and AnAOB. Even though COD presence resulted in the biodiversity increasing of AerAOB and decreasing of AnAOB, the dominant species were always Nitrosomonas and Candidatus brocadia during the whole experiment. Clone-sequencing of 16S rRNA results suggested the emergence of five different denitrifying species, which then led to a higher nitrogen removal. Results in this study demonstrated that the applied start-up strategy was feasible for SNAD process treating low C/N ratio sewage.

Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors

Autotrophic NH4(+) removal has been extensively researched, but few studies have investigated alternative electron acceptors (for example, SO4(2-)) in NH4(+) oxidation. In this study, sulfate-reducing anaerobic ammonium oxidation (SRAO) and conventional Anammox were started up in upflow anaerobic sludge blanket reactors (UASBRs) at 36 (±0.5)°C and 20 (±0.5)°C respectively, using reject water as a source of NH4(+). SO4(2-) or NO2(-), respectively, were applied as electron acceptors. It was assumed that higher temperature could promote the SRAO, partly compensating its thermodynamic disadvantage comparing with the conventional Anammox to achieve comparable total nitrogen (TN) removal rate. Average volumetric NH4(+)-N removal rate in the sulfate-reducing UASBR1 was however 5-6 times less (0.03kg-N/(m(3) day)) than in the UASBR2 performing conventional nitrite-dependent autotrophic nitrogen removal (0.17kg-N/(m(3) day)). However, the stoichiometric ratio of NH4(+) removal in UASBR1 was significantly higher than could be expected from the extent of SO4(2-) reduction, possibly due to interactions between the N- and S-compounds and organic matter of the reject water. Injections of N2H4 and NH2OH accelerated the SRAO. Similar effect was observed in batch tests with anthraquinone-2,6-disulfonate (AQDS). For detection of key microorganisms PCR-DGGE was used. From both UASBRs, uncultured bacterium clone ATB-KS-1929 belonging to the order Verrucomicrobiales, Anammox bacteria (uncultured Planctomycete clone Pla_PO55-9) and aerobic ammonium-oxidizing bacteria (uncultured sludge bacterium clone ASB08 "Nitrosomonas") were detected. Nevertheless the SRAO process was shown to be less effective for the treatment of reject water, compared to the conventional Anammox.

Increased salinity improves the thermotolerance of mesophilic nitrification

Nitrification is a well-studied and established process to treat ammonia in wastewater. Although thermophilic nitrification could avoid cooling costs for the treatment of warm wastewaters, applications above 40 °C remain a significant challenge. This study tested the effect of salinity on the thermotolerance of mesophilic nitrifying sludge (34 °C). In batch tests, 5 g NaCl L(-1) increased the activity of aerobic ammonia-oxidizing bacteria (AerAOB) by 20-21 % at 40 and 45 °C. For nitrite-oxidizing bacteria (NOB), the activity remained unaltered at 40 °C, yet decreased by 83 % at 45 °C. In a subsequent long-term continuous reactor test, temperature was increased from 34 to 40, 42.5, 45, 47.5 and 50 °C. The AerAOB activity showed 65 and 37 % higher immediate resilience in the salt reactor (7.5 g NaCl L(-1)) for the first two temperature transitions and lost activity from 45 °C onwards. NOB activity, in contrast to the batch tests, was 37 and 21 % more resilient in the salt reactor for the first two transitions, while no difference was observed for the third temperature transition. The control reactor lost NOB activity at 47.5 °C, while the salt reactor only lost activity at 50 °C. Overall, this study demonstrates salt amendment as a tool for a more efficient temperature transition for mesophilic sludge (34 °C) and eventually higher nitrification temperatures.

Structural Basis of Biological NO Generation by Octaheme Oxidoreductases

Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.

Structure and composition of biofilm communities in a moving bed biofilm reactor for nitritation-anammox at low temperatures.

It is a challenge to apply anaerobic ammonium oxidation (anammox) for nitrogen removal from wastewater at low temperatures. Maintenance of anammox- and aerobic ammonia oxidizing bacteria (AOB) and suppression of nitrite oxidizing bacteria (NOB) are key issues. In this work, a nitritation-anammox moving bed biofilm pilot reactor was operated at 19-10°C for 300 d. Nitrogen removal was decreasing, but stable, at 19-13°C. At 10°C removal became unstable. Quantitative PCR, fluorescence in situ hybridization and gene sequencing showed that no major microbial community changes were observed with decreased temperature. Anammox bacteria dominated the biofilm (0.9-1.2 × 10(14) 16S rRNA copies m(-2)). Most anammox bacteria were similar to Brocadia sp. 40, but another smaller Brocadia population was present near the biofilm-water interface, where also the AOB community (Nitrosomonas) was concentrated in thin layers (1.8-5.3 × 10(12) amoA copies m(-2)). NOB (Nitrobacter, Nitrospira) were always present at low concentrations (<1.3 × 10(11) 16S rRNA copies m(-2)).

Effects of allylthiourea, salinity, and pH on ammonia/ammonium-oxidizing prokaryotes in mangrove sediment incubated in laboratory microcosms

Anaerobic ammonium-oxidizing (anammox) bacteria, aerobic ammonia-oxidizing archaea (AOA) and bacteria (AOB) are three groups of ammonia/ammonium-oxidizing prokaryotes (AOPs) involved in the biochemical nitrogen cycling. In this study, the effects of allylthiourea (ATU), pH, and salinity on these three groups from mangrove sediment were investigated through microcosm incubation in laboratory. ATU treatments (50, 100, and 500 mg L(-1)) obviously affected the community structure of anammox bacteria and AOB, but only slightly for AOA. ATU began to inhibit anammox bacteria growth slightly from day 10, but had an obvious inhibition on AOA growth from the starting of the study. At 100 mg L(-1) of ATU or higher, AOB growth was inhibited, but only lasted for 5 days. The pH treatments showed that acidic condition (pH 5) had a slight effect on the community structure of anammox bacteria and AOA, but an obvious effect on AOB. Acidic condition promoted the growth of all groups of AOPs in different extent, but alkaline condition (pH 9) had a weak effect on AOB community structure and a strong effect on both anammox bacteria and AOA. Alkaline condition obviously inhibited anammox bacteria growth, slightly promoted AOA, and slightly promoted AOB in the first 20 days, but inhibited afterward. Salinity treatment showed that higher salinity (20 and 40 ‰) resulted in higher anammox bacteria diversity, and both AOA and AOB might have species specificity to salinity. High salinity promoted the growth of both anammox bacteria and AOB, inhibited AOA between 5 and 10 days, but promoted afterward. The results help to understand the role of these microbial groups in biogeochemical nitrogen cycling and their responses to the changing environments.

Performance and microbial community of completely autotrophic nitrogen removal over nitrite (CANON) process in two membrane bioreactors (MBR) fed with different substrate levels

To study the influence of substrate on completely autotrophic nitrogen removal over nitrite (CANON) process, two membrane bioreactors (MBR) with identical setup but fed with different substrate levels (R1 with low ammonia, R2 with high ammonia), were adopted in this study. The nitrogen removal performance, bioactivity, biodiversity and distribution of the functional microorganisms in two reactors were investigated. Both the aerobic ammonia-oxidizing bacteria (AerAOB) and anaerobic ammonia-oxidizing bacteria (AnAOB) in R2 showed higher bioactivity than those in R1, while nitrite-oxidizing bacteria (NOB) showed the contrary result. Nitrosomonas and Candidatus Kuenenia stuttgartiensis were detected as predominant functional microbes in the two reactors while Nitrobacter only existed in R1. High influent ammonia possibly led to the higher biodiversity of AerAOB and the more densely packed distribution. Meanwhile, this study has demonstrated the feasibility of increasing ammonia for rapid start-up, and decreasing HRT for high-rate nitrogen removal in CANON process.

Application of membrane bioreactor for completely autotrophic nitrogen removal over nitrite (CANON) process

A membrane bioreactor (MBR) was adopted for completely autotrophic nitrogen removal over nitrite (CANON) process at ambient temperature. CANON was rapidly started-up within around 50d under oxygen-limited condition. The average nitrogen removal rate reached to 0.70kgNm−3d−1 with a removal efficiency of 88%. The ratio of air flow rate to volumetric ammonium loading rate should be maintained below 0.28Lairmin−1kg−1Nm3d for stable CANON. The feasibility of MBR for CANON process was proved in batch experiments. FISH results showed that aerobic ammonium-oxidizing bacteria predominated in the reactor sludge, whereas anaerobic ammonium-oxidizing bacteria predominated in the membrane biofilm. This study demonstrated that MBR was a suitable experimental setup for the operation of CANON process.

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal.

Autotrophic nitrogen removal from domestic sewage in MBR–CANON system and the biodiversity of functional microbes

The feasibility of completely autotrophic nitrogen removal over nitrite (CANON) process for treating domestic sewage was investigated in membrane bioreactor (MBR), for which conventional activated sludge was seeded at ambient temperature. By gradually decreasing hydraulic retention time under the oxygen-limited condition, CANON was successfully started-up for 78 days. Finally the MBR-CANON system was adopted for treating domestic sewage, nitrogen and COD removal achieved to 0.97 kg m(-3) d(-1), 80%, respectively, with the effluent turbidity below 1.0 NTU. DGGE profiles showed a distinct community shift of the functional bacteria after seeded to the reactor, and phylogenetic results indicated the predominance of Nitrosomonas and Candidatus Kuenenia stuttgartiensis for nitrogen removal in the reactor. FISH results showed the predominance of aerobic ammonia oxidizing bacteria (AerAOB) and anaerobic ammonia oxidizing bacteria (AnAOB) in the system, both of whose proportion reduced when treated domestic sewage.